CN109295119B - Biocatalysis method for producing statin drug intermediate - Google Patents
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Abstract
The present application provides a biocatalytic method for the production of statin intermediate a7 comprising the steps of: taking tert-butyl (3R,5S) 6-chloro-3, 5-dihydroxyhexanoate, namely D3, as a substrate, wherein the substrate D3 is fed by 20-150g/L at one time; using halohydrin dehalogenase as a catalyst; adding a substrate NaCN aqueous solution continuously according to the pH of the reaction solution, wherein the pH of the reaction solution is controlled in a stepwise manner, and the pH is increased from the initial pH of 7.0 to the pH of 9.0; tert-butyl alcohol with the final concentration of 0.2-1.5 v/v% is added as a cosolvent and a hydrolysis inhibitor of D3 and A7, and the mixture is reacted for 1.5-4h at the temperature of 25-45 ℃ to finally obtain the product, namely the intermediate A7 of the atorvastatin. The present invention provides an effective solution to the hydrolysis problem of the A7 biocatalytic process to increase the productivity and A7 yield of the catalytic process.
Description
Technical Field
The invention relates to the technical field of medicine preparation, in particular to a biocatalysis method for producing statin medicine intermediate (3R,5R) 6-nitrile-3, 5-dihydroxy tert-butyl hexanoate (A7).
Background
The cardiovascular and cerebrovascular diseases are serious threats to human beings, have the characteristics of high morbidity, high disability rate and high mortality, and lead the number of people dying from the cardiovascular and cerebrovascular diseases to reach 1500 thousands of people every year all over the world to live at the head of various causes of death. In 2015, the total market of the national lipid-regulating drugs exceeds 200 million yuan RMB, the statins occupy the great majority, and the terminal retail price market of the national statins reaches 151.43 million yuan, which accounts for nearly 80% of the whole blood fat-reducing drug market. The clinical results of the statins on the market for 20 years show that the statins have high safety and small adverse reaction and are superior to other statins in the treatment of reducing LDL cholesterol. In 2015, the total market of Chinese statins reaches 83.19 billion yuan, which is 7.97% higher than that of the last year, and still shows a trend of rising year by year.
With the increasing market competition and the implementation of the social responsibility of enterprises, the production technology of atorvastatin needs to be improved and innovated urgently, so that the damage of industrial production to environmental resources and the waste of energy sources need to be reduced, and the enterprise cost and the drug cost of patients can be reduced, so that the economic benefit and the social benefit of the whole industry are improved. The key intermediate synthesis technology of the statin drugs is upgraded by adopting the biocatalysis technology, and a new energy-saving, green and efficient production process can be established. The halohydrin dehalogenase has a chemical synthesis step of replacing high energy consumption and high solvent consumption by a one-step conversion method, has the characteristic of high-efficiency catalysis, and is suitable for establishing a novel statin side chain synthesis process with low energy consumption, low cost and environmental protection.
One of the key difficulties of A7 biocatalysis is hydrolysis of products and substrates in aqueous solution, and Chinese patent application CN105567655A discloses a halohydrin dehalogenase and application thereof in synthesis of statin intermediates, wherein the document improves yield to a certain extent by recombining the halohydrin dehalogenase, but no patent publication solves the hydrolysis problem of A7 and D3 in the biocatalysis process. In some published patents, the time of biocatalytic conversion is more than 10 hours, during which hydrolysis of a7 and D3 is difficult to avoid effectively, how to reduce reaction time effectively and provide catalytic conversion efficiency is also the key to biocatalysis of a 7. The invention discloses a corresponding solution aiming at the two key problems of A7 biocatalysis, namely the hydrolysis problem of a product and a bottom and how to rapidly complete a catalytic reaction.
Disclosure of Invention
In view of the above problems with the prior art, the present application provides a biocatalytic method for the production of statin intermediates. The present invention provides an effective solution to the hydrolysis problem of the A7 biocatalytic process to increase the productivity and A7 yield of the catalytic process.
The technical scheme of the invention is as follows:
a biocatalytic process for the production of statin intermediate a7, said a7 being tert-butyl (3R,5R) 6-cyano-3, 5-dihydroxyhexanoate, comprising the steps of:
taking tert-butyl (3R,5S) 6-chloro-3, 5-dihydroxyhexanoate, namely D3, as a substrate, wherein the substrate D3 is fed by 20-150g/L at one time; using halohydrin dehalogenase as a catalyst; adding a substrate NaCN aqueous solution in a flowing manner according to the pH of the reaction solution, wherein the pH of the reaction solution is controlled in a stepwise manner, and is increased from the initial pH of 7.0 to the pH of 9.0; tert-butyl alcohol with the final concentration of 0.2-1.5 v/v% is added as a cosolvent and a hydrolysis inhibitor of D3 and A7, and the mixture is reacted for 1.5-4h at the temperature of 25-45 ℃ to finally obtain the product, namely the intermediate A7 of the atorvastatin.
The pH value of the reaction solution is controlled by increasing the pH value of the reaction solution by 0.2-0.5 every 10-30min from the initial pH value of the reaction solution, namely, the pH value of the reaction solution is increased in a stepped manner; the pH value of the reaction solution is increased by feeding alkaline NaCN aqueous solution.
Preferably, the mass concentration of the NaCN aqueous solution is 5-30%.
The catalytic reaction liquid is added with a cosolvent and a hydrolysis inhibitor tert-butyl alcohol of A7 and D3 in a mode that the tert-butyl alcohol is added at one time when the reaction starts.
The halohydrin dehalogenase is a halohydrin dehalogenase which catalyzes a substrate D3 to synthesize A7, and is a commercial enzyme preparation or enzyme fermentation broth; the material feeding amount is as follows: the dosage of the halogenated alcohol dehalogenase preparation is not less than 500U per kilogram of the substrate D3, and the halogenated alcohol dehalogenase preparation is fed once in the early stage of the reaction.
The product A7 is obtained by the catalytic reaction, the catalytic yield of A7 is not less than 95%, and the residual rate of D3 is not more than 3%.
Further, the substrate D3 is preferably added to a final concentration of 80 to 100g/L in the volume of the reaction liquid; the NaCN is preferably added in the form of a 10-30% NaCN aqueous solution by mass.
Further, the catalyst is a halohydrin dehalogenase having biological activity to catalyze the conversion of D3 to a7, including commercial halohydrin dehalogenase enzyme preparations or halohydrin dehalogenase fermentation broths prepared according to the published application patent (application No. CN 201810133923.2).
Further, the pH value of the reaction liquid is increased in a stepwise manner, namely the reaction of catalyzing D3 to convert A7 to generate hydrochloric acid, the pH value is reduced, and the pH value is automatically controlled by feeding alkaline NaCN aqueous solution as shown in figure 1. The pH value of the initial reaction liquid is controlled to be 7.0, and the pH value control point is increased by 0.2-0.5 every 10-30min after the reaction is started, namely the pH value of the reaction liquid is increased in a stepped manner.
Further, the final concentration of the added dissolution accelerator and the hydrolysis inhibitor, t-butanol, is preferably 0.5% (v/v) based on the volume of the reaction solution.
Further, the reaction temperature is 25 to 45 ℃ (preferably 35 to 40 ℃).
The beneficial technical effects of the invention are as follows:
in the biocatalysis process for producing the statin drug intermediate A7, a substrate, an intermediate product and a product are easy to hydrolyze, and the catalytic reaction yield is reduced, which troubles the production problem of high-efficiency production catalysis, and a related effective solution is not seen before. The method for preparing A7 by using (3R,5S) 6-chloro-3, 5-dihydroxy caproic acid tert-butyl ester (D3) as a substrate and commercial halohydrin dehalogenase enzyme preparation or enzyme fermentation broth as a catalyst and adopting a biocatalysis technology provides an effective solution to the hydrolysis problem of the A7 biocatalysis process, and is used for improving the productivity of the catalytic process and the yield of A7.
The main innovation points of the invention are embodied in two aspects:
firstly, according to the characteristics of acid production in the middle process of catalytic reaction and low stability of substrates D3 and A7 in aqueous solution, the invention designs a control method for stepwise increasing the pH of a reaction solution; the pH value of the reaction solution is increased in a stepwise manner by feeding an alkaline substrate NaCN aqueous solution, the catalytic reaction rate is accelerated, the reaction time is obviously reduced, the hydrolysis of the substrate and the product is reduced, and the catalytic conversion yield of A7 is effectively improved;
secondly, according to the structural characteristics of the substrate and the product and the characteristic of low water solubility, the tertiary butanol with double functions of promoting the substrate D3 to dissolve and inhibiting the hydrolysis of D3 and A7 is added, so that the reaction rate is effectively accelerated, and the hydrolysis of the product and the substrate is inhibited.
Through the process improvement of the two new methods, the reaction rate of D3 biocatalytic conversion A7 is improved by more than 20%, and the yield of the product A7 is improved by more than 15%. The biological catalysis yield of A7 is improved by 10-25%, and the biological catalysis production efficiency of A7 is obviously improved.
Drawings
FIG. 1 shows the reaction of halohydrin dehalogenase to catalyze the formation of A7 from substrate D3.
FIG. 2 is a gas chromatography analysis chart of the substrate D3, the intermediate product epoxide and the product A7 and a data analysis chart thereof at the initial stage and the later stage of the catalytic reaction.
FIG. 3 is a graph of the effect of different pH control points on the rate and conversion of D3 catalytic conversion A3.
FIG. 4 is a comparison of catalytic processes for single point pH control and gradient lift pH control.
FIG. 5 is a graph showing the effect of adding varying concentrations of a solubilizing agent and the hydrolysis inhibitor, tert-butanol, on the production of A7.
Detailed Description
The invention will be described in more detail with reference to the following figures and examples, but the scope of the invention is not limited thereto.
Example 1: biocatalytic reaction of A7 with different pH control points
Preparing a halohydrin dehalogenase fermentation broth: according to the published patent application (application No. CN 201810133923.2), a halohydrin dehalogenase fermentation broth is prepared, the cell density of the broth is 100g/L, and the enzyme activity of the dehalogenase is 160U/mL (enzyme activity definition and enzyme activity determination method, see the patent of application No. CN 201810133923.2).
Taking 10ml of the halogenated alcohol dehalogenase fermentation liquor, adding 30ml of distilled water, homogenizing under high pressure to obtain fresh halogenated alcohol dehalogenase enzyme liquid, and placing in a water bath at 40 ℃. And a stirring device and a pH automatic control device are installed. 4g of substrate D3 were added, i.e.the input was 100 g/L. The pH of the reaction solution was automatically titrated with a 10 wt% NaCN aqueous solution. Different pH control points were set, including pH7.0, pH7.5, pH8.0, pH8.5 and pH 9.0.
Sampling and analyzing during the reaction process. The sample treatment method is as follows: a sample (100. mu.L) was taken and added to 1.0mL of ethyl acetate; after sufficient shaking, centrifuging at 10000 Xg for 1 min; taking 100 mu L of supernatant, and adding the supernatant into 900 mu L of ethyl acetate; the diluted sample was used for gas chromatography.
The gas chromatographic analysis method is as follows: capillary chromatographic column: DB 170130 m × 0.53mm × 1.5 μm; column temperature: heating to 200 deg.C at 100 deg.C at 20 deg.C/min, and maintaining for 5 min; sample inlet temperature: 140 ℃; detector temperature: 260 ℃; carrier gas (N2): 5 ml/min; the split ratio is as follows: 20: 1; sample introduction amount: 1 mul; blank solution: and (3) ethyl acetate. During the reaction, chromatographic peaks of D3, the cyclized product and A7 are shown in FIG. 2.
The catalytic reaction process, adopting different pH automatic control points, has significant influence on the catalytic reaction rate and the A generation yield, and the comparison of the dynamic changes of D3 and A7 in the reaction process is shown in figure 3.
When the pH of the reaction solution was controlled to 7.0, the reaction rate was slow, and 72.3% of D3 remained after 10 hours of the reaction, whereas only 13.6% of A7 was produced. However, the decrease was slower with the total concentration of D3+ A7+ cyclates, indicating that these compounds are relatively stable at pH 7.0.
When the pH of the reaction solution was controlled to 7.5, the reaction rate was significantly improved as compared with that at pH7.0, and after 10 hours of reaction, 45.1% of D3 remained, whereas 44.6% of A7 was produced. While the total concentration decreased.
When the pH of the reaction solution was controlled to 8.0, the reaction rate was significantly improved as compared with that at pH7.5, and 9.8% of D3 remained and 80.6% of A7 was produced after 10 hours of reaction. While the total concentration decreased significantly.
When the pH of the reaction solution was controlled to 8.5, the reaction rate was fast, 5% of D3 remained after 6 hours of reaction, while 90.7% of A7 resulted, indicating that higher pH significantly increased the catalytic rate. But the total concentration is obviously reduced, and when the reaction is carried out for 3.5 hours, the total concentration is reduced by 24 percent compared with the total concentration when the reaction is carried out for 0.5 hour.
When the pH of the reaction solution is controlled to be 9.0, the reaction rate is fast, 3.2% of D3 is remained after the reaction is carried out for 3.5 hours, and 92.2% of A7 is generated, which shows that the higher pH can obviously improve the catalytic rate, but the total concentration is obviously reduced, which shows that the hydrolysis of the substrate and the product is obvious.
Therefore, the reaction of converting D3 into A7 is catalyzed by adopting halohydrin dehalogenase, the reaction rate can be obviously improved by increasing the pH of the reaction liquid, but the hydrolysis rates of a substrate and a product are increased at the same time, and the higher yield of A7 is difficult to achieve by a single pH control point.
Example 2: a7 biocatalytic reaction is carried out by adopting programmed step-type increase of pH of reaction liquid
Taking 10mi of the above halohydrin dehalogenase fermented enzyme solution, adding 30ml of distilled water, homogenizing under high pressure to obtain fresh halohydrin dehalogenase enzyme solution, and placing in a water bath at 40 ℃. And a stirring device and a pH automatic control device are installed. 4g of substrate D3 were added, i.e.the input was 100 g/L. The pH of the reaction solution was automatically titrated with a 10 wt% NaCN aqueous solution.
Setting procedural program to increase pH: pH7.0, 10 min; pH7.5, 10 min; pH8.0, 20 min; pH8.25, 30 min; pH8.5, 30 min; pH8.8, 20 min; pH9.0, 10 min. And a single control method of pH8.85 was used as a control. The catalytic process samples were analyzed and the ratio of D3 and A7 was varied as well as the concentration varied as shown in FIG. 4.
When the reaction is controlled by a single high pH value, namely pH8.85, the reaction rate is faster, and the reaction time is 3.5 hours, the residual concentration ratio of D3 is 5%, and the concentration ratio generated by A7 is 91.8%, so that the rapid catalysis is realized. But the total concentration is obviously reduced, and the reduction speed of the total concentration is accelerated when the reaction lasts for 3.5 hours. After 2 hours of reaction, the rate of production of A7 slowed and the concentration decreased instead, indicating an increased rate of hydrolysis.
The yield analysis of A7 was performed on the catalytic reaction solution with single pH control: extracting the reaction solution for 2 times by using ethyl acetate with 3 times of volume; combining the ethyl acetate extracts obtained in the 2 times, and fixing the volume to 250 mL; the extract was diluted 30-fold with ethyl acetate and used for gas chromatography. As a result, it was found that 2.74g of A7 was produced and 0.17g of D3 remained. The theoretical yield of A7 was 3.84g, so the actual yield of A7 was: 71.2 percent.
When the stepwise pH increase control method is adopted, as shown in FIG. 4, the reaction rate is also high, 5.9% of D3 remains after 3.5 hours of reaction, and 89.6% of A7 is generated. The rapid decrease in total concentration was effectively controlled, indicating a significant reduction in the rate of hydrolysis of the product and substrate. In addition, the concentration of a7 remained continuously increasing throughout the reaction, indicating that a7 was continuously produced and inhibited its too rapid hydrolysis.
And (3) carrying out A7 yield analysis on the catalytic reaction solution with gradient pH control: extracting the reaction solution for 2 times by using ethyl acetate with 3 times of volume; combining the ethyl acetate extracts obtained in the 2 times, and fixing the volume to 250 mL; the extract was diluted 20-fold with ethyl acetate and used for gas chromatography. As a result, it was found that 3.55g of A7 was produced and 0.2g of D3 remained. The theoretical yield of A7 was 3.84g, so the actual yield of A7 was: 92.4 percent.
Example 3: adding a cosolvent and a hydrolysis inhibitor tert-butyl alcohol to carry out the biocatalytic reaction of A7
Taking 10ml of the above-mentioned halohydrin dehalogenase fermented enzyme solution, adding 30ml of distilled water, homogenizing under high pressure to obtain fresh halohydrin dehalogenase enzyme solution, and placing in water bath at 40 ℃. And a stirring device and a pH automatic control device are installed. 4g of substrate D3 were added, i.e.the input was 100 g/L.
Different amounts of tert-butanol (in volume concentration) were added: 0%, 0.2%, 0.5%, 1.0%, 1.5%.
The pH of the reaction solution was automatically titrated with a 10 wt% NaCN aqueous solution, and the procedure for increasing the pH was as follows: pH7.0, 10 min; pH7.5, 10 min; pH8.0, 20 min; pH8.25, 30 min; pH8.5, 30 min; pH8.8, 20 min; pH9.0, 10 min.
A comparison of the formation of A7 with different amounts of tert-butanol added is shown in FIG. 5. The results show that the addition of t-butanol is effective in increasing the production rate of a 7. When tert-butanol at 0%, 0.2%, 0.5%, 1.0% and 1.5% concentration by volume is added to catalyze the reaction for 3.5h, the concentration of A7 is 76.3g/L, 82.4g/L, 91.8g/L, 89.2g/L and 87.7 g/L; the residual ratios of D3 were: 4.2%, 2.5%, 1.1%, 0.9% and 0.8%. Therefore, the addition of 0.2-1.5% of tertiary butanol can improve the yield of A7 by 8-20.3%, improve the catalytic reaction rate of D3 by more than 15%, and obviously reduce the residual rate of D3.
Example 4: catalytic conversion of 20g/L D3 to A7 with addition of a cosolvent and a hydrolysis inhibitor, tert-butanol
Taking 10ml of the above-mentioned halohydrin dehalogenase fermented enzyme solution, adding 30ml of distilled water, homogenizing under high pressure to obtain fresh halohydrin dehalogenase enzyme solution, and placing in water bath at 40 ℃. And a stirring device and a pH automatic control device are installed. 0.8g of substrate D3 was added, i.e.the input was 20 g/L. Addition of tert-butanol (in volume concentration): 0.5 percent. The pH of the reaction solution was automatically titrated with a 20 wt% NaCN aqueous solution, and the procedure for increasing the pH was as follows: pH7.0, 5 min; pH7.5, 10 min; pH8.0, 10 min; pH8.25, 10 min; pH8.5, 10 min; pH8.8, 5 min. The catalytic reaction was terminated within 50 min.
And (5) finishing the reaction, and carrying out sample treatment and analysis. The sample treatment method is as follows: extracting twice by using ethyl acetate with the volume being 3 times that of the mixture; the extract is added with ethyl acetate to fix the volume to 250 ml; the constant volume sample solution was diluted 5 times with ethyl acetate for gas chromatography.
The analysis result shows that the yield of A7 is 0.73g, the residual quantity of D3 is 0.0065g, namely the yield of A7 is as follows: 95%, the residual rate of D3 was: 0.8 percent.
Example 5: catalytic conversion of 150g/L D3 to A7 with addition of a cosolvent and a hydrolysis inhibitor tert-butanol
Taking 10ml of the above-mentioned halohydrin dehalogenase fermented enzyme solution, adding 30ml of distilled water, homogenizing under high pressure to obtain fresh halohydrin dehalogenase enzyme solution, and placing in water bath at 40 ℃. And a stirring device and a pH automatic control device are installed. 6g of substrate D3 were added, i.e.the input was 150 g/L. Addition of tert-butanol (in volume concentration): 0.5 percent. The pH of the reaction solution was automatically titrated with a 30 wt% NaCN aqueous solution, and the procedure for increasing the pH was as follows: pH7.0, 10 min; pH7.5, 20 min; pH8.0, 30 min; pH8.25, 60 min; pH8.6, 60 min; pH8.8, 30 min; pH9.0, 30 min. The catalytic reaction was terminated within 4 h.
And (5) finishing the reaction, and carrying out sample treatment and analysis. The sample treatment method is as follows: extracting twice by using ethyl acetate with the volume being 3 times that of the mixture; the extract is added with ethyl acetate to fix the volume to 250 ml; the constant volume sample was diluted 50 times with ethyl acetate for gas chromatography.
The analysis result showed that the yield of A7 was 5.08, the residual amount of D3 was 0.12g, i.e., the yield of A7 was: 88%, the residual ratio of D3 was: 2.0 percent.
Claims (5)
1. A biocatalytic process for the production of statin intermediate a7, said a7 being (3R,5R) 6-cyano-3, 5-dihydroxyhexanoic acid tert-butyl ester, characterized in that it comprises the steps of:
taking tert-butyl (3R,5S) 6-chloro-3, 5-dihydroxyhexanoate, namely D3, as a substrate, wherein the substrate D3 is fed by 20-150g/L at one time; using halohydrin dehalogenase as a catalyst; adding a substrate NaCN aqueous solution in a flowing manner according to the pH of the reaction solution, wherein the pH of the reaction solution is controlled in a stepwise manner, and is increased from the initial pH of 7.0 to the pH of 9.0; adding tert-butyl alcohol with the final concentration of 0.2-1.5 v/v% as a cosolvent and a hydrolysis inhibitor of D3 and A7, and reacting for 1.5-4h at the temperature of 25-45 ℃ to finally obtain a product, namely an atorvastatin intermediate A7;
the pH value of the reaction solution is controlled by increasing the pH value of the reaction solution by 0.2-0.5 every 10-30min from the initial pH value of the reaction solution, namely, the pH value of the reaction solution is increased in a stepped manner; the pH value of the reaction solution is increased by feeding alkaline NaCN aqueous solution.
2. The method according to claim 1, characterized in that the mass concentration of the NaCN aqueous solution is 5-30%.
3. The method as claimed in claim 1, wherein the reaction mixture is added with t-butanol as a cosolvent and as a hydrolysis inhibitor of a7 and D3 in such a manner that the t-butanol is added at once at the beginning of the reaction.
4. The method of claim 1, wherein the halohydrin dehalogenase is a halohydrin dehalogenase having a catalytic substrate D3 to synthesize a7, being a commercial enzyme preparation or a fermentation broth of an enzyme; the material feeding amount is as follows: the dosage of the halogenated alcohol dehalogenase preparation is not less than 500U per kilogram of the substrate D3, and the halogenated alcohol dehalogenase preparation is fed once in the early stage of the reaction.
5. The process according to claim 1, characterized in that the catalytic reaction yields product a7, the catalytic yield of a7 being not less than 95% and the residual yield of D3 being not more than 3%.
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| US7632666B2 (en) * | 2004-12-27 | 2009-12-15 | Masarykova Univerzita V Brne | Method of production of optically active halohydrocarbons and alcohols using hydrolytic dehalogenation catalysed by haloalkane dehalogenases |
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| CN104651290A (en) * | 2015-02-10 | 2015-05-27 | 浙江大学 | Engineering bacterium and application thereof in preparation of atorvastatin drug intermediate |
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